Parkin protein as ubiquitin ligase

ABSTRACT

The use of Parkin protein (Parkin) as ubiquitin ligase (E3), and a new method of diagnosing juvenile Parkinsonism based on the use thereof.

BACKGROUND OF THE INVENTION

The present invention relates to a new use of Parkin protein(hereinafter referred to as “Parkin”). In particular, the presentinvention relates to a use of Parkin as ubiquitin ligase (E3). Thepresent invention also relates to a diagnosis method of juvenileParkinsonism on the basis of Parkin's ubiquitin ligase (E3) function. Inparticular, the present invention also relates to a diagnosis method ofjuvenile Parkinsonism by detecting a mutation of Parkin gene(hereinafter referred to as “parkin”). The present invention furtherrelates to a method of screening medicines for treating and/orpreventing juvenile Parkinsonism.

It is known that the ubiquitin pathway plays an important role in theintracellular protein decomposition which relates to the control of theamount of protein in the cells [Hershko, A et al, Annu. Rev. Biochem.67, 425-479 (1998); and Hochstrasser, M, Annu. Rev. Genet. 30, 405-439(1996)]. Ubiquitin is linked with a target protein through an isopeptidelink of C-terminal Gly of ubiquitin and ε-NH₂ group in Lys residue ofthe target protein. A protein is converted into ubiquitin in thepresence of three enzymatic cascade catalysts, i. e. E1 (ubiquitinactivation enzyme), E2 (ubiquitin linking enzyme) and E3 (ubiquitinligase). A polyubiquitin chain formed by the series of the reactionsfunctions as a decomposition signal for the proteolytic attack by 26Sproteasome which is an ATP-depending protease complex of eukaryotes[Coux O. et al., Annu. Rev. Biochem. 65. 801-847 (1996); and BaumeisterW., et al. Cell 92, 367-380 (1998)]. Because of the variety of E2(ubiquitin linking enzyme) and E3 (ubiquitin ligase), two or morepathways of the proteolysis in the cells are obtained [Hershko A. etal., Annu. Rev. Biochem. 67, 425-479 (1998); and Hochstrasser M., Annu.Rev. Genet. 30, 405-439 (1996)]. The details of the molecular base of E3which most closely relates to the substrate recognition have not yetbeen elucidated. It is suggested at present that various RING-fingerproteins relate, as E3 to the ubiquitination of proteins [Harper J. W.et al., Nature Cell Biol. 1, E5-E7 (1999); Zachariae W. et al., Genes &Dev. 13, 2039-2058 (1999): Xie Y. et al., EMBO J. 18, 6832-6844 (1999);Joazeiro C. A. P. et al., Science 286, 309-312 (1999); Lorick K. L. etal., Proc. Natl. Acad. Sci. USA 96, 11364-11369 (1999); Moynihan T. P.et al., J. Biol. Chem. 274, 30963-30968 (1999); and Martinez-Noel G. etal., FEBS Leters 454, 257-261 (1999)].

The brief description will be made on E1, E2 and E3.

E1 (ubiquitin activation enzyme) is an enzyme which first formsubiquitin molecules and a high-energy thioester link. ATP is consumed inthe formation of the high-energy thioester link with the ubiquitinmolecule. E1 transfers the activated ubiquitin to the following E2(ubiquitin linking enzyme).

E2 receives the activated ubiquitin from E1 to form a new thioesterintermediate. It was already found that many of E2 selectively interactwith E3 (ubiquitin ligase), some E2 directly ubiquitinate substrateproteins. E2 forms a gene family. 13 genes were identified in buddedyeast. It is now being elucidated that in higher animals such as humanbeings, a larger number of genes are present.

E3 (ubiquitin ligase) directly or indirectly interact with a substrateprotein (target protein) to catalyze a reaction of transferringubiquitin to the substrate or a ubiquitin chain already formed in thesubstrate. E3 is the most important functional molecule for thedetermination of the substrate specificity of the ubiquitin system.Number of E3 enzymes reported until now is only small, and the functionsof them have been scarcely elucidated. At present, E3 enzymes areclassified into two groups of HECT type and RING finger type. Therelationship between abnormality of E3 enzymes and diseases has beensuggested.

-   (1) HECT type: a family of E3 enzymes having HECT (homologous to    E6-AP at C-terminus) domain homologous to E6-AP (E6-associated    protein) at C-terminus; and-   (2) RING finger type: The following 6 RING finger types have already    been known:-   (2-1) E-3α (a homologue of UBR1 of yeast) and E3β which recognize    N-terminal of substrate (target)-   (2-2) Component APC11 of APC (anaphase promoting complex) having a    molecular weight of about 1,500 KDa effective mainly in a cell    division period,-   (2-3) ROC1 which is a linking factor of SCF [SkpI/Cullin 1    (Cdc53)/F-box protein] complex effective mainly in G1 period,-   (2-4) MDM2 which relates to the ubiquitination of p53,-   (2-5) Adapter protein c-Cb1 of receptor tyrosine kinase, and-   (2-6) Mammary cancer—causing gene product BRCA1.

Recently, some genes causative hereditary nerve degeneration diseaseswere identified, and the dynamics of abnormal proteins produced by thecausative genes is now being elucidated. Consequently, it was found thatmany abnormal proteins aggregate and deposit in the brains and nerveswhich are damaged by the diseases. Therefore, a common molecularmechanisms caused by the deposition of the abnormal proteins are assumedin the nerve degeneration. It is now tried to develop a new treatmentmethod by elucidating the mechanisms.

Parkinson's disease is a typical nerve degeneration disease mainlycharacterized by extrapyramidal symptoms. Pathological characteristicsof Parkinson's disease are that nigra dopamigenergic neurons and otherbrain stem neurons are degenerated and fall out and that Lewy bodiesappear in the cell bodies of remaining neurons. It was found recentlythat α-synuclein protein and ubiquitin are accumulated in the Lewybodies of neurons of patients. The missense variation of α-synucleingenes was also found, and it was suggested that this variation is acause of the abnormal intracellular accumulation.

Recently, genes causing autosomal recessive juvenile Parkinson's disease(AR-JP) of young human beings, which is of recessive inheritance, wereidentified after the linkage analysis, and they were named “parkin”[Kitada T. et al., Nature 392, 605-608 (1998)]. “parkin” was a new genederived from human No. 6 chromosome q 25.2-27. The protein (Parkin)encoded with parkin has a domain [ubiquitin-like (Ubl) domain]homologous to the ubiquitin at the N-terminal. Supposing Parkin concernsthe proteolysis like ubiquitin, losing of the function thereof causesaccumulation of some proteins (abnormal proteolysis) and injure theneurons. Parkin had a RING-box, comprising two RING finger domains andan IBR (in between RING) between them, at C-terminal. However, noevidence showing that Parkin concerns the ubiquitin pathway was reporteduntil now. The function of Parkin in the cells has not been fullyelucidated yet.

DISCLOSURE OF THE INVENTION

The object of the present invention is to find a new function of Parkinby examining whether Parkin concerns the ubiquitin path or not and thento provide a novel method of diagnosing juvenile Parkinsonism on thebasis of the finding. Another object of the present invention is toprovide a method for screening medicines for preventing and/or treatingjuvenile Parkinson's disease by using Parkin having a changed functionin the ubiquitin pathway.

After intensive investigations made for the purpose of solving theabove-described problems, the inventors have succeeded in proving thefact that Parkin concerns the transfer reaction of ubiquitin into thetarget protein in the ubiquitin pathway. The present invention has beencompleted on the basis of this finding.

The present invention provides the use of Parkin protein (Parkin) asubiquitin ligase (E3). In an embodiment of the present invention,ubiquitin ligase is an enzyme having an activity of linking with aubiquitin linking enzyme (E2). to link ubiquitin with a target protein.

In another aspect, the present invention provides a diagnosis method ofdetecting a variation in Parkin gene (parkin), i. e. a diagnosis methodof juvenile Parkinsonism by detecting a variation that Parkin exerts aninfluence on the activity of linking ubiquitin to a target protein inneurons and/or a variation which exerts an influence on the interactionof Parkin protein (Parkin) and ubiquitin linking enzyme (E2).

Preferably, the variation that exerts an influence on the activity oflinking ubiquitin to a target protein in neurons is the variation ordeletion in the ubiquitin-like (Ubl) domain of parkin gene (parkin)and/or RING-box or the neighborhood thereof.

Preferably, the variation which exerts an influence on the interactionbetween Parkin and ubiquitin linking enzyme (E2) is the variation ordeletion in the RING-box of Parkin gene (parkin) or the neighborhoodthereof.

The ubiquitin linking enzyme (E2) is preferably UbcH7 or UbcH8.

In still another aspect, the present invention provides a diagnosismethod of juvenile Parkinsonism which comprises determining an activityof linking ubiquitin with the target protein in neurons in Parkinprotein (Parkin) of the patient, and also a diagnosis method of juvenileParkinsonism which comprises determining the interaction between Parkinprotein (Parkin) of the patient with ubiquitin linking enzyme (E2).

The ubiquitin linking enzyme (E2) is preferably UbcH7 or UbcH8.

In still another aspect, the present invention provides a method ofscreening medicines for treating and/or preventing juvenileParkinsonism, which comprises:

-   (1) a step of providing cells having an expression vector containing    Parkin gene (parkin) from a patient with juvenile Parkinsonism,-   (2) a step of bringing the cells into contact with a candidate    compound, and-   (3) a step of determining the activity of Parkin protein (Parkin) to    link ubiquitin with a target protein and/or the interaction of    Parkin protein (Parkin) and a ubiquitin linking enzyme (E2).

The cells are preferably those derived from nerves.

In still another aspect, the present invention provides a medicine fortreating and/or preventing juvenile. Parkinsonism identified by thescreening method of the present invention.

In still another aspect, the present invention provides cells carryingan expression vector having parkin gene (parkin) obtained from patientswith juvenile Parkinsonism, which are to be used for the screeningmethod of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph showing the results of immunoblotting, whichshows the association of Parkin and UbcH7 in human fetal kidney 293cells.

FIG. 2 a is a schematic view showing the structure of Parkin and thestructure of artificial or natural variant of Parkin used in Example 2.FIG. 2 b is a photograph showing the results of the immunoblottingshowing the association of UbcH7 with varied Parkin.

FIG. 3 is a picture showing the results of the immunoblotting, whichshows the association of the ubiquitinated cell protein and Parkin afterthe treatment with MG132 in human dopamine neuroblastoma SH-SY5Y cell.

FIG. 4 is a picture showing the results of the immunoblotting, whichshows the results of the domain analysis of Parkin necessitated for thelinking with ubiquitinated protein.

FIG. 5 is a schematic view showing a model of ubiquitination pathway towhich Parkin relates. In FIG. 5, Ub indicates ubiquitin, E1 indicates aubiquitin activation enzyme, E2 represents a ubiquitin linking enzyme,Ub1 represents a ubiquitin-like domain, and “X” represents a targetprotein assumed to be recognized by the ubiquitin-like domain of Parkinfor the ubiquitination.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the use of Parkin protein (Parkin) asubiquitin ligase (E3). The embodiment of the term “use” of Parkin asubiquitin ligase (E3) is not particularly limited. It indicates the useof Parkin for the diagnosis of Parkinson's disease, particularlyjuvenile Parkinsonism, or the use thereof for screening medicines forthe diagnosis, prevention and/or treatment of Parkinson's disease,particularly juvenile Parkinsonism.

Ubiquitin ligase (E3) is an enzyme directly or indirectly interact witha target protein to transfer ubiquitin into a target protein. In somecases, a ubiquitin chain has already been formed in the target protein.In such a case, ubiquitin ligase (E3) catalyzes the reaction oftransferring ubiquitin into the already formed ubiquitin chain. Anexpression “Ubiquitin is linked with (or transferred into) a targetprotein” herein indicates that ubiquitin is transferred into thenon-ubiquitinated target protein per se or it is transferred into aubiquitin chain of already ubiquitinated target protein.

In some cases, it is necessary that ubiquitin ligase (E3) is linked withubiquitin linking enzyme (E2) for exhibiting its effect of linkingubiquitin to the target protein.

The present invention also relates to a method of diagnosing juvenileParkinsonism. In the diagnosis method of the present invention, avariation which exerts an influence on activity of Parkin for linkingubiquitin with the target protein in neurons and/or a variation whichexerts an influence on the interaction of Parkin and the ubiquitinlinking enzyme (E2).

Parkin is reported in Kitada T. et al, Nature 392, 605-608 (1998).“parkin” is composed of 12 exons and it encodes 465 amino acids.“parkin”has a ubiquitin-like domain (amino acid Nos. 1 to 76) atN-terminal, and RING1 domain (amino acid Nos. 238 to 293), IBR domains(amino acid Nos. 314 to 377) and RING2 domain (amino acid Nos. 418 to449) at C-terminal. The parkin gene variations of Japanese and Turkishpatients are analyzed. It is now being elucidated that in patients withjuvenile Parkinsonism, frequency of deletion of a specified exon ishigh. The term “RING box”herein indicates an amino acid sequence ofamino acid numbers of 238 to 449, and “around it” indicates about 10 to20 amino acid residues around the RING box.

It is detected in Northern blotting that mRNA of Parkin is generallyexpressed, and the expression in the brain is particularly high. As willbe shown in Examples given below, Parkin has an activity ofubiquitinating a target protein in human dopamigenergic neuroblastomaSH-SY5Y cells. The activity of Parkin to catalyze the ubiquitin linkingreaction is considered to be kept by certain cells including neurons.Therefore, it is preferred to use neurons such as human dopamigenergicneuroblastoma SH-SY5Y cells.

The variation which exerts an influence on the activity of linkingubiquitin with a target protein involves a variation of eitherdecreasing or increasing the activity of linking ubiquitin with thetarget protein. It is generally considered that when the linkage ofubiquitin with the target protein is reduced, the decompositionefficiency of the target protein is lowered and, as a result, theaccumulation of the protein is caused, though the present invention isnot limited by this theory. It is possible, therefore, that juvenileParkinsonism can be diagnosed by determining the lowering of theubiquitin linking activity.

The variation of Parkin which exerts an influence on the activity oflinking ubiquitin with the target protein is caused by variation orlacking of a base in one or more of ubiquitin-like domain, RING1 domain,IBR domain and RING2 domain of parkin. Such a variation can be detectedby directly determining the base sequence of DNA taken from a patient orit can be easily detected by PCR when the presence or absence of analready known variation is to be found.

The term “ubiquitin linking enzyme (E2)” herein indicates an enzymewhich receives activated ubiquitin from E1 to form a new thioesterintermediate. This enzyme is preferably UbcH7 or UbcH8. It isparticularly preferably UbcH7.

UbcH7 is a protein composed of 154 amino acids and having a molecularweight of 17861 Da (Ulrike Nubert et al., J. Biol. Chem., 271,2795-2800, 1996). UbcH8 is a protein composed of 153 amino acids andhaving a molecular weight of 17768 Da (Sushant Kumar et al., J. Biol.Chem., 272, 13548-13554, 1997).

In the ubiquitination of E6 dependent P53 derived from papilloma virusesof type 16 and type 18, E6-AP (100 kDa) having hectodomain functions asa ligase. It was reported that the ubiquitin linking enzymes (E2) inthis case are UbcH5, UbcH7 and UbcH8 (Sushant Kumar et al., J. Biol.Chem., 272, 13548-13554, 1997).

The homology of UbcH7 and UbcH8 is about 46%.

In the diagnosis method of the present invention, the activity of Parkinto link ubiquitin with a target protein in neurons and/or theinteraction of Parkin and ubiquitin linking enzyme (E2) is determined.

The method of determining the activity of Parkin to link ubiquitin withthe target protein in neurons is not limited. For example, an expressionvector containing parkin labeled with Myc or the like and a ubiquitinexpression vector labeled with FLAG or the like are simultaneouslytransfected into neurons such as SH-SY5Y cells. The cells are dividedinto 2 groups. The cells in one group are treated with a proteasomeinhibitor such as MG132. A certain period after the transfection, thecells are recovered. A ubiquitin-linked protein is detected by applyingthe immunoblotting method wherein an antibody (such as anti FLAGantibody) to the substance used for the labeling ubiquitin is used, toan immunoprecipitate prepared by using an antibody (such as antiMycantibody) to a substance used for the labeling Parkin. When ahigh-molecular ubiquitinated protein is detected by this method, it issuggested that ubiquitin is lined with the target protein. When a bandcorresponding to the high-molecular ubiquitinated protein is notdetected in the cells which were not treated with the proteasomeinhibitor, it is suggested that the high-molecular ubiquitinated proteinhas been decomposed with the proteasome. Thus the concern of Parkin withthe proteolysis can be examined.

In the diagnosis method of the present invention, the interactionbetween Parkin and ubiquitin linking enzyme (E2) is examined. Theinteraction can be determined by any method. For example, an expressionvector containing parkin labeled with Myc or the like can be transfectedinto a proper cell together with that containing a gene encoding aubiquitin-linked enzyme labeled with FLAG, His, HA or the like. Acertain period after the transfection, a cell extract is prepared andthe immunoprecipitation is conducted with an antibody (such as antiMycantibody) against the label used for labeling Parkin. The interactionbetween Parkin and ubiquitin linking enzyme (E2) can be detected by theimmunoblotting with the immunoprecipitate and an antibody against thelabel used for labeling the ubiquitin linking enzyme.

The present invention also provides a method of screening a medicine fortreating and/or preventing juvenile Parkinsonism. The screening methodof the present invention comprises the following steps:

-   (1) a step of providing cells keeping an expression vector    containing parkin from a patient with juvenile Parkinsonism,-   (2) a step of bringing the cells into contact with a candidate    compound, and-   (3) a step of determining the activity of Parkin to link ubiquitin    with a target protein and/or the interaction between Parkin and    ubiquitin linking enzyme (E2).

The expression vectors used in the present invention are vectors capableof expressing insertion genes preferably with animal cells, particularlypreferably with human neurons. The expression vectors are preferablythose capable of autonomous replication in host cells or capable ofintegration into chromosomes and containing a promoter at a position atwhich the inserted gene can be transcribed. The kinds of the expressionvectors are not particularly limited and, for example, pcDNA3.1(+)vector (Invitrogen), p-Tet-On vector (Clontech), pSI MammalianExpression Vector (Promega) and pSVK3 expression vector (AmershamPharmacia Biotech) are usable.

The kinds of the cells usable in the present invention are notparticularly limited so far as Parkin can exhibit its function as aubiquitin ligase in the ubiquitin pathway. The cells are, however,preferably animal cells, particularly neurons and more particularlyhuman neurons such as human dopamigenergic neuroblastoma.

When a candidate compound improves the activity of Parkin to linkubiquitin with a target protein or it improves the interaction betweenParkin and ubiquitin linking enzyme (E2), the candidate compound can beselected as a candidate for a medicine for treating and/or preventingjuvenile Parkinsonism. The medicine thus selected can be subjected tothe subsequent tests to confirm the medicinal effect thereof.

The kinds of the candidate compounds are not particularly limited, andthey include, for example, cytokines, low-molecular medicines, hormones,specificity antibodies, peptide imitations, antisense oligonucleotidesand other medicines capable of altering the cell functions or proteinexpression.

The following Examples will further illustrate the present invention,which by no means limit the invention.

EXAMPLE Experiment Operation

(1) Expression Plasmid and Transfection

For preparing pcDNA3.1(+)Myc and pcDNA3.1(+)FLAG vectors, oligo DNAencoding N-terminal Myc and FLAG epitopes was ligated at KpnI/BamHIdomain of pcDNA3.1(+) (Invitrogen). After the amplification of wild typeparkin or N-terminal-deficient variant, Ubc2(HHR6B), UbcH8 and cDNA ofubiquitin with a suitable primer, they were ligated in BamHI domain ofthe vector. The C-terminal-deficient variant and missense variant ofparkin were prepared by introducing a variation intopcDNA3.1(+)Myc-Parkin by using a Quik Change domain specific variationinducing kit (Stratagene) according to a manual. pCAGGS-Ubc3 was alreadyreported [Iwai K. et al., Proc. Natl. Acad. Sci. USA 96, 12436-12441(1999)].

pCGN-Ubc4 and other pcDNA3.1(+)FLAG-Ubc were prepared from human livercDNA library by an ordinary method. Ubc used was obtained from humanbeings in all the cases. Human fetal kidney 293 (HEK293) cells and humandopamigenergic neuroblastoma SH-SY5Y cells were cultured in DMEMcontaining 10% bovine fetal serum, 10 μg/ml streptomycin and 100 U/mlpenicillin.

The transfection was carried out by using FuGENE6 transfection reagentaccording to the producer (Boehringer-Mannheim)'s manual. The cells weretreated with MG132 (Cbz-Leu-Leu-Leu-aldehyde) (Peptide Institute, Inc.,Osaka, Japan) having a final concentration of 50 μM.

(1) Immunological Analysis

The immunoprecipitation was conducted with rabbit polyclonal antiMycantibodies (A-14) (Santa Cruz Biotechnology) as previously reported[Suzuki H. et al., Biochem. Biophys. Res. Commun. 256, 127-132 (1999)].The immunoblotting was conducted with biotin linked mouse monoclonalantiFLAG(M2) antibody (SIGMA), mouse monoclonal antiMyc antibody (9E10)(Santa Cruz Biotechnology), mouse monoclonal antiHA antibody (HA.11)(Berkeley Antibody Company) or mouse monoclonal antiHis antibody(RGS.His) (QIAGEN). Streptoavidin/horseradish peroxidase complex(Amersham Pharmacia Biotech) was used for detecting biotin-linkedantiFLAG(M2) antibody.

Example 1 Interaction Between Parkin and E2 Enzyme

The following test was conducted to find whether Parkin concerns theubiquitin pathway or not. To find whether the interaction between Parkinand each of various E2 enzymes occurs or not, Myc-labeled parkin wasexpressed together with various E2 enzymes labeled with FLAG, HA or Hisat different domains in HEK293 cells, and the interaction of them wasdetected by the immunoprecipitation The results are shown in FIG. 1.

Concretely, pcDNA3.1(+)Myc-Parkin (10 1 μg) was transfected in HEK293cells simultaneously with various expression vectors encoding FLAG-,His- or HA-Ubc in an amount shown in the parentheses in FIG. 1. 48 hoursafter the transfection, cell extracts were prepared, and theimmunoprecipitation (IP) was conducted with antiMyc antibody. The cellextracts (upper panel in FIG. 1) and immunoprecipitates (middle andlower panels in FIG. 1) were analyzed by the immunoblotting with variousantibodies as shown in FIG. 1. Although the association of Parkin andUbcH8 was not detected under ordinary conditions, it was detected when alarge amount of UbcH8 was expressed and the immunoblot films wereexposed for a long period of time for ECL reaction (refer to # lane).Symbol (*) indicates a nonspecific band including IgG light chain.

As shown in FIG. 1, UbcH7 and UbcH8 immunoprecipitated together withParkin (UbcH8 immunoprecipitation was weak). However, no detectablelinkage was found in other tested E2 enzymes (such as Ubc2, Ubc3, Ubc4,UbcH5A-C and UbcH6).

Example 2

The domain structure of Parkin which can interact with UbcH7 wasanalyzed. Parkin has 3 domains, namely ubiquitin-like domain atN-terminal, RING-box domain at C-terminal and linker domain which linksthese two segments. It is known that the RING-box domain at C terminalis composed of RING1, RING2 and IBR (in-between-RING) [Morett E. et al.,Trends Biochem. Sci. 24, 229-231 (1999)] as shown in FIG. 2 a. FIG. 2 ais a schematic view showing the structures of the artificial and naturalvariants of Parkin used in Example 2. Amino acid is represented by oneletter notation in FIG. 2 a. Nonsense isomer and missense isomer ofParkin found in patients with juvenile Parkinsonism (AR-JP) are shown asParkin^(Q311stop) and Parkin^(T240R)/Parkin^(R42P). Parkin^(Q311stop) isa variant formed by replacing No. 211 glutamine with stop codon, andParkin^(T240R)/Parkin^(R42P) is a variant formed by replacing No. 240threonine with arginine and No. 42 arginine with proline.

The interaction between variant Parkin and UbcH7 was analyzed by usingthe variant Parkin in the same manner as that of Example 1 except thatthe amount (μg) of each variant Parkin was as shown in parentheses inFIG. 2 b, and pcDNA3.1(+)FLAG-UbcH7 (7 μg) was used. In theimmunoblotting wherein antiFLAG antibody was used, the crude extract wasused (upper panel in FIG. 2 b). In the immunoblotting wherein antiMycantibody (middle panel in FIG. 2 b) or antiFLAG antibody (lower panel inFIG. 2 b) was used, the immunoprecipitate obtained with antiMyc antibodywas used. Symbol (*) shows the heavy chain and light chain of IgG.

It is understood from the results shown in FIG. 2 b that the deletion ofubiquitin-like domain and linker domain exerted no influence on thelinkage with UbcH7 and that only a half of C-terminal having RING-boxwas enough for the complement of UbcH7. On the contrary, RING-boxvariant lacking in RING1 or RING2 domain could not be linked with UbcH7.In the tests of two missense variants of patients with juvenileParkinsonism (AR-JP), Parkin^(R42P) which was a ubiquitin-like domainvariant could be linked with UbcH7, while Parkin^(T240R) in which Thrwas changed to Arg [Hattori N. et al., Biochem. Biophys. Res. Commun.249, 754-758 (1998)] did not interact with UbcH7 in RING1 domain. Fromthese results, it was found that Ring-box domain (not other domains suchas the linker domain or ubiquitin-like domain) was necessary for theassociation with UbcH7 which is a ubiquitin linking enzyme.

Example 3

Tests were conducted to know whether Parkin can catch a target proteinto ubiquitinate it as reported for E3 of other classes so as toelucidate the roles of Parkin in ubiquitin pathway [ZacHAriae W. et al.,Genes & Dev. 13, 2039-2058 (1999); Xie Y. et al., EMBO J. 18, 6832-6844(1999); Joazeiro, C. A. P. et al. Science 286, 309-312 (1999); andLorick K. L. et al. Proc. Natl. Acad. Sci. USA 96, 11364-11369 (1999)].

At first, 10 μg of pcDNA3.1(+)Myc-parkin and 10 μg ofpcDNA3.1(+)FLAG-ubiquitin vector were simultaneously transfected intoSH-SY5Y cells. 48 hours after the transfection, all the cells wererecovered. In one of the experiments, the cells were treated with MG132(50 μM) 3, 8 or 24 hours before the recovery of the cells. Animmunoprecipitate prepared with antiMyc antibody was used for theimmunoblotting with antiFLAG antibody (upper panel in FIG. 3) andantiMyc antibody (lower panel in FIG. 3). In FIG. 3, a symbol (*)represents the position of Parkin. A ubiquitinated protein of a highmolecular weight is shown as (Ub)n on the right side.

As will be understood from the results shown in FIG. 3, when Myc-parkinand FLAG-ubiquitin were simultaneously expressed with humandopamigenergic neuroblastoma SH-SY5Y cells, ubiquitinated bandsincluding high-molecular ubiquitinated proteins, were detected withantiMyc immunoprecipitates from a crude extract of cells which had beentreated with a proteasome inhibitor MG132 (Cbz-Leu-Leu-Leu-aldehyde)[Rock K. L. et al., Cell 78, 761-771 (1994)]. On the other hand, whenthe treatment with an inhibitor was not conducted, no ubiquitinated bandwas found. The association of ubiquitinated labeled protein with Parkinproceeded depending on the time after the treatment of MG132. Some ofthe ubiquitinated proteins were smaller than Parkin. These resultsindicated that Parkin can catch the cell proteins for the ubiquitinationand that it can function as ubiquitin protein ligase.

In the same analysis as above but wherein HEK293 fetal kidney cellextract was used, no ubiquitinated band linked with Parkin was observed(no data are shown). These results suggested that target protein ofParkin was lacking in HEK293 cells or another indispensable factor whichexerts an influence on the linkage of Parkin with the target protein oron the E3 activity of Parkin is lacking in HEK293 cells and thatubiquitination-inhibiting protein is contained in HEK293 cells.

Example 4

It was tried to elucidate the functional relationship between thelinkage of Parkin and E2 in SH-SY5Y neurons and the ubiquitinationactivity.

The experimental conditions were the same as those of Example 3. Theexpression vectors of various variants Myc-Parkin were used in an amount(μg) given in parentheses in FIG. 4. Before recovering the cells, theywere treated with MG132 (50 μM) for 24 hours. 48 hours after thetransfection, the immunoprecipitate prepared with antiMyc antibody wasused for the immunoblotting with antiFLAG antibody (upper panel in FIG.4) and antiMyc antibody (lower panel in FIG. 4). The details of thevariant Parkin are shown in FIG. 2 a. In FIG. 4, a symbol (*) representsthe heavy chain and light chain bands of IgG as in FIG. 2 b. The band ofIgG heavy chain overlaps with the positions of wild Parkin and missensevaried Parkin.

As shown in FIG. 4, when a lack or missense variation occurred inRING-box, which made the uptake of UbcH7 impossible, a band of theubiquitinated protein associated with Parkin was not detected under thesame conditions of treatment with MG132. These results indicated thatthe uptake of UbcH7 is indispensable for the function of Parkin. Parkinvariants lacking in ubiquitin-like domain and varied Parkin^(R42P) takenfrom a patient substantially completely lost the association with theubiquitinated protein (FIG. 4). However, because these varied Parkincould be linked with UbcH7 (FIG. 2 b), it was suggested that theubiquitin-like domain is indispensable for the recognition of thetarget. These results suggested that the loss in the ubiquitinationactivity of Parkin is a cause of AR-JP.

Discussion of Examples

It was suggested that Parkin concerns juvenile Parkinson syndrome whichcauses nerve degeneration of nigra [Kitada T. et al., Nature 392,605-608 (1998); and Mizuno Y. et al., J. Neurochem. 71, 893-902 (1998)].The results of Examples 1 to 4 suggested that Parkin functions as aligase (E3 enzyme) for linking ubiquitin with a target protein withUbcH7 (E2 enzyme) and that it catalyzes the ubiquitination of the targetprotein. FIG. 5 is a schematic view showing the mechanism. Parkin iscomposed of two functionally different domains, namely, (1) C-terminalRING-box for catching UbcH7 which is a specific E2 enzyme and (2)N-terminal ubiquitin-like domain necessary for the recognition of targetprotein for the ubiquitination. Many AR-JP patients have deficiencyvariation of Parkin and some of the patients have missense variation inRING-box or ubiquitin-like domain [Hattori N. et al., Biochem. Biophys.Res. Commun. 249, 754-758 (1998); and Hattori N. et al., Ann. Neurol.44. 935-941 (1998)]. In our tests, all the Parkin constructions having avariation in this domain or lacking this domain were lacking in E3activity. This fact indicates that the ubiquitination pathway mediatedby Parkin plays an important role in keeping the homeostasis of neuronof nigra by controlling the amount of protein.

However, the mechanism of the fact that dysfunction of Parkin induces ahighly selective death of neurons is still unknown. It was found inexperiments according to the present invention that ubiquitinatedprotein is not immunoprecipitated in HEK293 cells of fetal kidneys,while it is immunoprecipitated together with Parkin in SH-SY5Y cells innerves. This finding suggests that Parkin selectively participates inthe proteolysis in neurons. When Parkin is regarded to be ubiquitinligase, a factor indispensable for the recognition of the substrate witha target protein (shown as “X” in FIG. 5) and/or Parkin might be presentin only dopamigenergic neurons. Possibly, an abnormal accumulation of“X” in the nigra cells of patients with AR-JP induces a specific deathof neurons. This phenomenon can probably be explained by the findingthat Parkin concerns the ubiquitination and it would act as ubiquitinprotein ligase.

Evidences proving the fact that the abnormal accumulation ofubiquitinated protein is often observed in various nerve degenerationdiseases are increasing more and more [Lowe J. et al., Brain Pathol. 3,55-65 (1993); and Mayer R. et al. (1998), In “Ubiquitin and the Biologyof the Cell” pp. 429-462, plenum Press, New York]. These findingsindicate that the control in amount of protein intermediated byubiquitin/proteasome pathway plays an important role in manynon-dividing neurons.

Before the present invention, it was unknown that the variation ofparkin in patients with AR-JP is related to the abnormality of theubiquitination.

The function of parkin has been partially elucidated and a new diagnosismethod for juvenile Parkinsonism was provided by the present invention.Also the development of a screening system for medicines usable for theprevention and/or treatment of Parkinson's disease was made possible bythe present invention.

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 14. A method of diagnosing juvenile Parkinsonism, saidmethod comprising: detecting a variation in a Parkin gene, wherein saidvariation exerts an influence on activity of linking ubiquitin with atarget protein in neurons or exerts an influence on interaction ofParkin protein and ubiquitin linking enzyme (E2).
 15. The method ofclaim 14, wherein the variation occurs in a ubiquitin-like (Ubl) domainor RING-box of the Parkin gene or RING-box and exerts an influence onactivity of linking ubiquitin with a target protein in neurons.
 16. Themethod of claim 14, wherein the variation occurs in a RING-box of Parkingene and exerts an influence on the interaction between Parkin proteinand ubiquitin linking enzyme (E2).
 17. The method of claim 14, whereinthe ubiquitin linking enzyme (E2) is UbcH7 or UbcH8.
 18. The method ofclaim 15, wherein the ubiquitin linking enzyme (E2) is UbcH7 or UbcH8.19. The method of claim 16, wherein the ubiquitin linking enzyme (E2) isUbcH7 or UbcH8.
 20. A method of diagnosis of juvenile Parkinsonism, saidmethod comprising: determining an activity of linking ubiquitin with atarget protein in neurons in Parkin protein of a patient.
 21. A methodof diagnosis of juvenile Parkinsonism, said method comprising aninteraction between Parkin protein of a patient with an ubiquitinlinking enzyme (E2).
 22. The method of diagnosis according to claim 21,wherein the ubiquitin linking enzyme (E2) is UbcH7 or UbcH8.
 23. Amethod of screening for a compound useful for treating or preventingjuvenile Parkinsonism, said method comprising: (a) providing a candidatecompound to cells having an expression vector containing Parkin genefrom a patient with juvenile Parkinsonism; and (b) determining activityof Parkin protein to link ubiquitin with a target protein or determininginteraction between Parkin protein and a ubiquitin linking enzyme (E2),thereby determining whether the candidate compound is useful fortreating or preventing juvenile Parkinsonism.
 24. The method of claim23, wherein the cells are derived from nerves.